PROJECT SUMMARY The long-term goal of this project is to understand how epithelial stem cells in the skin establish distinct patterns of gene activation and silencing during their differentiation into specialized cell lineages and how these genetic programs are re-organized during skin regeneration and tumorigenesis. It is now widely accepted that in addition to signaling/transcription factor-mediated mechanisms, lineage- specific gene expression programs are also regulated epigenetically, i.e., via covalent DNA/histone modifications, as well as via spatial arrangements of the genes and their enhancer elements in the nucleus. Enhancer-promoter interactions serve as key determinants providing functional and structural frameworks for cell-specific transcription controlled by lineage-specific transcription factors. However, enhancer-promoter regulatory networks are substantially re-organized during cell transition towards malignancy, which include an aberrant exposure of gene promoters to inappropriate regulatory elements resulting in activation of pro- oncogenes, as well as in silencing of the tumor suppressor genes. CCCTC-binding factor (CTCF) serves as one of the core architectural proteins that plays a key role in the control the establishment and maintenance of enhancer/promoter interactions. CTCF gene is frequently mutated in cancers, and abrogation of its tumor-suppressor activity contributes to cancer development and progression. Our preliminary data demonstrate that CTCF is expressed in both mouse and human epidermis, while Krt14-driven Ctcf genetic ablation results in severe alterations in skin development, epidermal barrier maintenance, and tumorigenesis. In this multi-PI proposal, we will test a hypothesis that CTCF serves as critical determinant that control the establishment and maintenance of the enhancer-promoter regulatory networks in skin epithelial stem cells and their progenies during development and postnatal homeostasis, while alterations in such networks result in skin tumorigenesis. We will address this hypothesis via two Specific Aims: 1. Define the roles of CTCF in the establishment and maintenance of lineage-specific higher-order chromatin structure, enhancer-promoter regulatory networks and gene expression in distinct populations of skin epithelial cells during development and hair cycle-associated skin regeneration. 2. Delineate the roles for Ctcf in regulation of gene expression, 3D chromatin organization and enhancer-promoter interactions in the skin epithelial cells during tumorigenesis. This project will have a fundamental impact on our current knowledge of epigenetic mechanisms that regulate genome reorganization in stem cells during their differentiation and tumorigenesis and will promote the progress towards the development of novel epigenetic drugs as a new paradigm for treatment of skin disorders.